The present invention relates to the field of somatic embryo production, particularly to methods for somatic embyrogenesis of Jatropha from ovules. More specifically, the present invention relates to a method and media compositions for somatic embryogenesis of Jatropha curcas from ovules of unopened flower buds. The method is well suited for Jatropha curcas transformation, for producing clonal planting stock useful for large scale Jatropha curcas plantation and for producing haploids, double haploids, diploids and disease-free plantlets.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the Bibliography.
Jatropha curcas, belonging to the family of Euphorbiaceous, is a plant of Latin American origin, widely spread throughout the arid and semi-arid tropical regions of the world. Jatropha is a large genus comprising over 170 species. The most common species in India are J. curcas, J. glandulifera, J. gossypifolia, J. multifida, J. nana, J. panduraefolia, J. villosa and J. podagrica. J. curcas is a small tree or shrub with smooth gray bark, which exudes whitish colored, watery, latex when cut. Normally, it grows between three and five meters in height, but can attain a height of up to eight or ten meters under favorable conditions. It is a drought-resistant plant, living up to 50 years and growing on marginal lands.
J. curcas has large green to pale green leaves, which are aligned alternate to sub-opposite. The leaves are three-five lobed with a spiral phyllotaxis. The petiole of the flowers ranges between 6-23 mm in length. The flowers are formed in hot seasons. Several crops are formed provided the soil is moisture is good and temperatures are high. In conditions where continuous growth occurs, an imbalance of pistillate or staminate flower production results in a higher number of female flowers. Fruits are produced in winter when the shrub is leafless. Each inflorescence yields a bunch of approximately 10 or more ovoid fruits. Three, bi-valved cocci are formed after the seeds mature and the fleshy exocarp dries. The seeds become mature when the capsule changes from green to yellow, after two to four months from fertilization. The blackish, thin-shelled seeds are oblong and resemble small castor seeds. This plant has various medicinal uses especially in nutraceuticals, pharmaceutical, dermatological, and personal care products. The latex of Jatropha curcas has anticancer properties due to the presence of an alkaloid known as “jatrophine.” The tender twigs are used for cleaning teeth. The juice of the leaf is used for external application for piles. The roots are used as an antidote for snake-bites. The seeds are used for antihelmithic purposes. The bark yields a dark blue dye used for coloring cloth, fish net and lines. Most of the Jatropha species are ornamental except for J. curcas and J. glandulifera which are oil-yielding species. The seeds of these species contain semi-dry oil which has been found useful for medicinal and veterinary purposes.
The oil content is 25-30% in the seeds and 50-60% in the kernel. The oil contains 21% saturated fatty acids and 79% unsaturated fatty acids. Jatropha oil contains linolenic acid (C 18:2) and oleic acid (C18:1) which together account for up to 80% of the oil composition. Palmitic acid (C16:0) and stearic acid (C18:0) are other fatty acids present in this oil. The oil is non-edible, however it has the potential to provide a promising and commercially viable alternative to diesel oil as it has all the desirable physicochemical and performance characteristics as that of diesel. The plant J. curcas has lately attracted particular attention as a tropical energy plant. The seed oil can be used as a diesel engine fuel for it has characteristics close to those of fossil fuel diesel. Moreover, due to its non-toxic and biodegradable nature, Jatropha biodiesel meets the European EN 14214 standards of a pure and blended automotive fuel for diesel engines. Jatropha curcas seed yields approach 6-8 MT/ha with ca 37% oil. Such yield could produce the equivalent of 2100-2800 liters of fuel oil/ha, whose energy is equivalent to 19,800-26,400 kwh/ha (Gaydou et al., 1982). Because of its very high saponification value and its ability to burn without emitting smoke, the oil of the seeds is commercially useful. For example, it is extensively used for making soap. The current economic, environmental and energy security concerns worldwide forces to shift from fossil fuels to biofuel alternatives such as bioethanol and biodiesel. Since biofuels can be produced from a diverse set of crops, each country is adopting a strategy that exploits the comparative advantages it holds in certain crops.
Among the different biofuel crops, Jatropha curcas is considered to be a better candidate for the production of biofuel because of the physiological and natural factors. The intense interest in oil from Jatropha curcas has generated enormous pressure to supply enough seeds that are homogenous and productive enough for plantation. Therefore, there is an urgent need to mass propagate elite trees. Equally urgent are methods to improve various agronomical traits of Jatropha curcas. Genetic engineering is recognized as a fast method for crop improvement. Plant transformation is essentially a two step process, i.e., delivery of genes into a host cell followed by regeneration of the transformed cell into a plant. Somatic embryogenic calli or somatic embryogenic suspension cultures is generally regarded as the most efficient method of regeneration as most of the transformed cells have already acquired the embryogenic potential that will drive them to develop into a somatic embryo quite spontaneously, a process similar to a fertilized egg cell in a zygotic embryo (Dodeman, et al., 1997).
Somatic embryos are suitable for transformation via Agrobacterium tuniefaciens (Mathews et al., 1992), microinjection (Neuhaus et al., 1987) and particle bombardment (Wilde et al., 1992). In addition, somatic embryos or somatic embryogenic calli can be cryopreserved using liquid nitrogen without loss of viability. This they are ideal materials for maintenance of germplasm as well as cell embryogenecity.
Somatic embryos are clonal in origin and thus multiplication using somatic embryos can have the potential for exceedingly high rates of vegetative increase and is therefore of considerable commercial interest. Regeneration via somatic embryogenesis is an attractive option for plant tissue culture. Somatic embryos reportedly provide more stable regenerants than shoots. Another advantage of regeneration systems using somatic embryos is their apparent single cell origin. This means that it is unlikely that regenerants are of chimerical origin, since, if a regenerant originates from a cluster of cells rather than a single sell, the plant tissues may be chimerical or unstable and produce off-types.
To further improve this crop, biotechnological techniques such as tissue culture and transformation can be utilized. Over two decades several reports were documented on Jatropha regeneration using various explants using different composition of media. These reports include plant regeneration from hypocotyl, petiole, and leaf explants (Sujatha and Mukta, 1996), epicotyl explants (Chinese Patent Application No. 200610020449.X), leaf disc (Indian Patent Application Publication No. 490/MUM/2006), shoot tip and nodal explants (European Patent Application Publication No. 1817956; Datta et al., 2007), somatic embryogenesis from leaf callus (Jha et al., 2007), and transformation from cotyledon disc explants (Li et al., 2008). All of the above studies focused on callus-mediated and meristematic tissue regeneration. Production of plantlets via organogenesis or somatic embryogenesis from reproductive tissues (anthers or ovules) may lead to the production of haploids, double haploids or diploids which may result in true to type homozygous character, paying the way from plant breeding program.
Ploidy determinations have traditionally been done by counting chromosomes of stained root tips, but this method is laborious and often difficult with species which have small chromosomes and high ploidy levels and can lead to misclassified germplasm (Brummer et al., 1999). All chromosomes are located in the cell nucleus of plants enabling nuclear DNA content to be used as an estimate of ploidy level. In recent years; flow cytometry has become the preferred technique for estimating the nuclear DNA content because of its ease, quickness, and accuracy (Rayburn et al., 1989; Heslop-Harrison, 1995). Arumuganathan and Earle (1991a) determined nuclear DNA contents of more than 100 major crop plant species using flow cytometry. Vogel et al. (1999) used flow cytometry to determine the base DNA content of the genomes in the perennial Triticeae. Flow cytometry also has been used to determine the ploidy level of switchgrass (Panicum virgatum L.) (Hultquist et al., 1997; Lu et al., 1998), alfalfa (Medicago sativa L.) (Brummer et al., 1999) and turfgrass species (Arumuganathan et al., 1999). The amount of DNA in plant cells is expressed in picograms (pg) as a “C” value (Bennett and Smith, 1976). The letter C stands for a “constant” or the amount of DNA in a haploid nucleus or genome; 2C values represent the DNA content of a diploid somatic nucleus. DNA amounts in picograms can be converted to megabase pairs (Mbp) by means of the conversion factor of 1 pg=980 Mbp (Bennett et al., 2000).
Therefore, there remains a need in the art for regeneration of plantlets via somatic embryogenesis using reproductive tissues (anthers or ovules). Despite these reported success, in this investigation we report for the first time, high frequency regeneration via somatic embryogenesis from ovule explants isolated from unopened flower buds of Jatropha curcas which are economical and allow production of haploids, double haploids or diploids and disease-free plantlets.